1. Field of the Invention
Having established the feasibility of producing a wide variety of naturally occurring and synthetic polypeptides by means of hybrid DNA technology, there are continuing and extensive efforts to provide for more efficient and economic methods for producing the polypeptides. In developing a process for the commercial production of polypeptides, many factors will be involved in optimizing the economic and efficient production of the polypeptides. Included among these factors are regulatory signals, which are DNA sequences involved with the regulation of replicaton, transcription and translation.
One area of interest is at the level of transcription. Transcription involves the enzyme RNA polymerase. The RNA polymerase binds to a site called a promoter. It has been observed that promoters vary in their activity, as evidenced by the number of initiations of RNA per unit time or the strength of binding of the enzyme to the promoter site. The promoter may have one or more sequences that bind, which may or may not be contiguous. The more active promoters are referred to as strong promoters.
It was found that when introducing a strong promoter into a vector and employing the resulting plasmid for transformation, one could not select transformants based on expression of markers which allowed for selection. Therefore, cloning of the strong promoters was not feasible. It is therefore desirable that methods be provided which would allow for the screening of strong promoters and terminators and their subsequent cloning to be used in conjunction with the replication, transcription and translation of the genes for production of DNA, RNA, and polypeptides.
2. Description of the Prior Art
Promoters from bacterial and viral sources have been cloned in E. coli, and their signal strength in vitro has been studied using expression from distal promoterless sequences encoding .beta.-galactosidase or other proteins (Casadaban and Cohen (1980) J. Mol. Biol 138, 179-207; West and Rodriguez (1980) Gene 9, 175-193). Attempts to clone small DNA fragments carrying the strong promoters of bacteriophage T5 have been unsuccessful (v. Gabain and Bujard (1979) PNAS USA 76, 189, 193), Fragments of T5 DNA having both a strong promoter and a strong termination signal have been cloned. (Breunig (1979) Dissertation (Universitat Heidelberg, Heidelberg, Germany)) Analysis has shown that transcriptional regions of several E. coli plasmids are organized in units where initiation and termination signals, are balanced. (Stuber and Bujard (1981) PNAS USA 78: 167-171) P.sub.25 and P.sub.26 promoters of the T5 bacteriophage are reported as among the most efficient RNA polymerase binding sequences. (Stuber et al (1978) Mol. gen. Genet. 166 141-149; Niemann ( 1981) Diplomarbeit (Universitat Heidelberg, Heidelberg, Germany)).